Chemokines are small, secreted pro-inflammatory proteins, which mediate directional migration of leukocytes from the blood to the site of injury. Depending on the position of the conserved cysteines characterizing this family of proteins, the chemokine family can be divided structurally into C, CC, CXC and CX3C chemokines that bind to a series of membrane receptors (Baggiolini M et al., 1997; Fernandez E J and Lolis E, 2002). These membrane receptors, all heptahelical G-protein coupled receptors, allow chemokines to exert their biological activity on the target cells, which may present specific combinations of receptors according to their state and/or type. The physiological effects of chemokines result from a complex and integrated system of concurrent interactions: the receptors often have overlapping ligand specificity, so that a single receptor can bind different chemokines. A single chemokine can bind to different receptors as well.
Studies on structure-activity relationships indicate that chemokines have two main sites of interaction with their receptors, the flexible amino-terminal region and the conformationally rigid loop that follows the second Cysteine. Chemokines are thought to dock onto receptors by means of the loop region, and this contact is believed to facilitate the binding of the amino-terminal region that results in receptor activation.
Usually, chemokines are produced at the site of injury and cause leukocyte migration and activation, playing a fundamental role in inflammatory, immune, homeostatic, hematopoietic, and angiogenic processes. Thus, these molecules are considered good target candidates for therapeutic intervention in diseases associated with such processes. The inhibition of chemokines, or of their receptors, can reduce leukocyte maturation, recruitment and activation, as well as other pathological processes related to angiogenesis or arteriosclerosis (Baggiolini M, 2001; Loetscher P and Clark-Lewis I, 2001; Godessart N and Kunkel S L, 2001).
In addition to mutant inhibitory chemokines, antibodies and peptide and small molecule inhibitors blocking the receptors the search for effective chemokine antagonists has also been extended to a series of viruses and other organisms that, when entering into contact with human or mammal hosts, show potent immunomodulatory activities affecting the host.
The viral mimicry of cytokines, chemokines, and their receptors may indicate strategies of immune modulation for developing therapeutic products (Alcami A, 2003; Lindow M et al., 2003). Recently, immunomodulatory factors expressed by haematophagous arthropods (such as mosquitoes, sandflies and ticks) have been reviewed (Gillespie, R D et al., 2000; Nuttall P A et al., 2000; Schoeler G B and Wikel S K, 2001).
In particular, the salivary glands of ticks produce a complex mixture of bioactive molecules having, in particular, anti-inflammatory, anti-haemostatic and anti-immune activities. These include bioactive proteins that control histamine, bind immunoglobulins, or inhibit the alternative complement cascade or other proteases.
The effect of these molecules is, probably, to provide a privileged site at the tick-host interface that shelters the tick from the normal innate and acquired host immune mechanisms that combat infections, ensuring successful feeding.
Moreover, tick salivary glands are considered the major route by which tick-borne pathogens enter the host during feeding, since ticks use their salivary glands as a means of concentrating the blood meal by returning the excess fluid and ions back to the host, possibly transmitting pathogens resident in these glands. In fact, tick induced modulation of host immunity is increasingly recognized as an important factor in successful transmission or establishment of tick-borne pathogens.
Immunomodulating activities have been characterized in tick saliva extracts (Alarcon-Chaidez F J et al., 2003; Bergman D K et al., 2000; Anguita J et al., 2002; Gwakisa P et al., 2001; Leboulle G et al., 2002; Kopecky J et al., 1999; Kovar L et al., 2002; Gillespie R D et al., 2001). For example, the saliva from Rhipicephalus sanguineus inhibits antigen-stimulated production of immunoglobulins and the expression of IFN-gamma, IL-2 and IL-5 in a dose-dependent manner (Matsumoto K et al., 2003).
CXC-chemokine binding activities, in particular CXCL8/Interleukin 8 binding activities, have been detected (but not characterized in terms of specific protein sequences) in the saliva prepared from several ixodid tick species (Dermacentor reticulatus, Amblyomma variegatum, Rhipicephalus appendiculatus, Haemaphysalis inermis, Ixodes ricinus), demonstrating a reduction of the level of detectable IL-8, and inhibiting IL-8 induced chemotaxis of human blood granulocytes. (Hajnicka V et al., 2001; Kocakova P et al. 2003; WO 01/58941; WO 01/48484).
Antigens from Rhipicephalus sanguineus elicit potent cell-mediated immune responses in resistant but not in susceptible animals. The saliva introduced during tick infestations reduces the ability of a susceptible animal host to respond to tick antigens that could stimulate a protective immune response. As a consequence, the animals present a disturbed cellular migration to the tick feeding site, which can represent a deficient response against ticks (Ferreira B R et al., 2003).
A homologue of the pro-inflammatory cytokine Macrophage Migration Inhibitory Factor has been detected in the tick, Amblyomma americanum. This sequence inhibited the migration of human macrophages in an in vitro functional assay to the same extent as recombinant human MIF (Jaworski D C et al., 2001; WO 01/78770).
Despite the large amount of literature, only a few articles list cDNA sequences identified by random sequencing and differential screens of libraries generated from various tick tissues and/or species. Lists of cDNA sequences have been published for Amblyomma americanum and of Dermacentor andersoni at different developmental stages (Hill C A and Gutierrez J A, 2000), salivary glands of unfed and fed male Amblyomma americanum (Bior A D et al., 2002), male mating Ixodes scapularis (Packila M and Guilfoile P G, 2002), salivary glands of Amblyomma variegatum (Nene V et al., 2002), of Rhipicephalus appendiculatus (Nene V et al., 2004), and of Ixodes scapularis (Valenzuela J G et al., 2002a; Francischetti I M et al., 2002).
However, the large majority of these sequences are not characterized biochemically or functionally, and many annotations are entered only on the basis of sequence similarity with known proteins involved in basic cellular functions, such as those previously characterised in tick salivary glands for enzymatic activities or inducing antibody response. In particular, there is no indication of tick proteins acting as CC-chemokine binding proteins.